RESEARCH REPORT

CORONA, PHABULOSA and PHAVOLUTA collaborate withBELL1 to confine WUSCHEL expression to the nucellus inArabidopsis ovulesToshihiro Yamada1,*, Yusuke Sasaki1, Kayo Hashimoto2,3, Keiji Nakajima2 and Charles S. Gasser4

ABSTRACTAngiosperm ovules consist of three proximal-distal domains – thenucellus, chalaza and funiculus – demarcated by developmental fateand specific gene expression. Mutation in three paralogous class IIIhomeodomain leucine zipper (HD-ZIPIII) genes leads to aberrationsin ovule integument development. Expression of WUSCHEL (WUS)is normally confined to the nucellar domain, but in this triplemutant expression expands into the chalaza. MicroRNA-inducedsuppression of this expansion partially suppresses the effects of theHD-ZIPIII mutations on ovule development, implicating ectopic WUSexpression as a component of the mutant phenotype. bell1 (bel1)mutants produce aberrant structures in place of the integuments andWUS is ectopically expressed in these structures. Combination ofbel1 with the HD-ZIPIII triple mutant leads to a striking phenotype inwhich ectopic ovules emerge from nodes of ectopicWUS expressionalong the funiculi of the primary ovules. The synergistic phenotypeindicates that BEL1 and the HD-ZIPIII genes act in at least partialindependence in confining WUS expression to the nucellus andmaintaining ovule morphology. The branching ovules of the mutantresemble those of some fossil gymnosperms, implicating BEL1 andHD-ZIPIII genes as players in the evolution of the unbranched ovuleform in extant angiosperms.

KEY WORDS: Ovule, HD-ZIPIII, WUS, BEL1, Chalaza, Integument,Arabidopsis thaliana

INTRODUCTIONOvules are the developmental precursors of seeds. In angiosperms,the ovule consists of three developmental domains; the nucellus,chalaza and funiculus (Fig. 1A) (Balasubramanian and Schneitz,2000). The nucellus contains a megasporangium, where the femalegametophyte develops. The inner and outer integuments form fromthe chalaza and enclose the nucellus. The funiculus is a stalk-likestructure that connects the ovule to the ovary wall. For correctdevelopment of the ovules, it is essential to establish this proximal-distal patterning; hence, a shift in the boundary between domainsresults in aberrant ovule morphology (e.g. Balasubramanian andSchneitz, 2000; Gross-Hardt et al., 2002).WUSCHEL (WUS) is a homeobox gene that characterizes the

nucellus by its restricted expression in this domain, and this nucellar

expression is necessary for initiation of the two integuments thatdevelop from the chalaza (Gross-Hardt et al., 2002).WUS promotesthe expression of an auxin efflux facilitator, PIN-FORMED 1(PIN1), via transcriptional activation of SPOROCYTELESS [SPL;also known as NOZZLE (NZZ)], and this mechanism is necessaryfor nucellus formation (Bencivenga et al., 2012). Extension of theinteguments is incomplete when WUS expression is driven in thechalzal domain by the AINTEGUMENTA promoter ( pANT) (Gross-Hardt et al., 2002). In pANT≫WUS plants additional putative outerinteguments also emerge just below the extended WUS expressionarea (Gross-Hardt et al., 2002; Sieber et al., 2004), as if a newboundary is established between the nucellus and chalaza. Theseresults indicate thatWUS regulation is key to defining the nucellus-chalaza boundary.

A recent study showed that externally applied cytokinin expandsWUS expression into the chalaza (Bencivenga et al., 2012). WUSexpression is also altered in bell1 (bel1) (Bencivenga et al., 2012;Brambilla et al., 2007) (see Fig. S2), but the change in theexpression is not as profound, suggesting that other factors playroles in WUS regulation.

Class III homeodomain leucine zipper (HD-ZIPIII) genesestablish the identity of the adaxial tissue of lateral organs (Emeryet al., 2003; McConnell et al., 2001), as well as regulating WUSexpression in shoot and floral apices in cooperation with CLAVATA3or ERECTA (ER) homologs (Green et al., 2005; Landau et al., 2015;Lee and Clark, 2015; Mandel et al., 2014). In ovules, HD-ZIPIIIgenes are involved in the development of the integuments; thus, inmost ovules of loss-of-functionHD-ZIPIII genemutants [i.e. coronaphabulosa phavoluta (cna phb phv)] the integuments are absent,reduced or malformed (Kelley et al., 2009). The mechanism of theseeffects remains unclear.

Here,we show thatCNA,PHB andPHV cooperatively repressWUSexpression in the chalaza, and that this repression is important forovule development. A combination of thesemutationswith bel1 leadsto additional misexpression of WUS outside of the nucellus and anovel phenotype not seen in either class of mutant. Thus, we identifytranscription factors necessary for boundary demarcation between thenucellus and chalaza and for patterning ofWUS expression.

RESULTS AND DISCUSSIONWUS expression extends into the chalaza in cna phb phvIn cna phb phv carpels with ovules at stage 2 (for stages, seeSchneitz et al., 1995),WUS expression was∼1.8-fold higher than inwild-type (WT) carpels at the same stage (Fig. S1). Sincegynoecium formation is almost completed in cna phb phv at thisstage, we hypothesize that the increase results from elevated WUSexpression in developing ovules.

In contrast to WT, where WUS expression was confined to thenucellus (Fig. S2), in most cna phb phv ovules at stage 2-II, WUSReceived 14 August 2015; Accepted 11 December 2015

1School of Natural System, College of Science and Engineering, KanazawaUniversity, Kanazawa 920-1192, Japan. 2Graduate School of Biological Sciences,Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. 3Graduate School of Humanities and Sciences, Nara Women’sUniversity, Nara 630-8506, Japan. 4Department of Molecular and Cellular Biology,University of California, Davis, CA 95616, USA.

*Author for correspondence (ptilo@mb.infoweb.ne.jp)

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expression extended into the chalaza, including the integuments(Fig. 1B). Misexpression ofWUS in the chalazawas still observed inovules at stage 2-III, whereas expression in the nucellus decreased(Fig. 1C). A subset of ovules did, however, exhibit WT-likeexpression of WUS (Fig. 1D). In cna phb phv some ovules dodevelop normally, although most exhibit aberrant integumentdevelopment (Kelley et al., 2009). Coexistence of ovules withnormal and abnormal WUS expression is thus consistent with theovule phenotypes.In summary, CNA, PHB and PHV are required for preventing

WUS expression in the chalaza. However, the sporadic WT-likephenotype in cna phb phv ovules implies that other gene(s) are alsoinvolved in the regulation of WUS.

Ovule defects in cna phb phv are suppressed bypCNA:amiRWUSGrowth of the integuments was disturbed in pANT≫WUS plants(Gross-Hardt et al., 2002). Thus, the misexpression of WUS couldaccount for the aberrant shape of the integuments in cna phb phv.The ectopically expressed WUS in cna phb phv was knocked

down using an artificial microRNA for WUS (amiRWUS). Sincethe CNA promoter ( pCNA) drives gene transcription throughout thechalaza, including the two integuments, we utilized pCNA as thedriver of amiRWUS expression (Fig. S1). In gynoecia of cna phb phvpCNA:amiRWUS at stage 2, amiRWUS reducedWUS expression to a

level that averaged 60% of that observed in cna phb phv (Fig. 2A).Suppression of WUS expression was also evaluated using a gWUS-GFP3 transgene (Tucker et al., 2008) (Fig. S2). This gene showed anexpanded GFP signal in cna phb phv, but the expanded signal wasabsent from the chalaza in almost all ovules of cna phb phv pCNA:amiRWUS (compare Fig. 2C,E with 2D,F; Fig. S2).

Development of the integuments was restored in most ovules ofcna phb phv pCNA:amiRWUS (compare Fig. 2G,I with 2H,J). Thepercentage of normal ovules per carpel significantly increased incna phb phv pCNA:amiRWUS as compared with cna phb phv(Fig. 2B). Therefore, the misexpression ofWUS partly accounts forthe aberrant shape of the integuments in cna phb phv.

Similar to phenotypes of loss-of-function cna phb phv mutants,ovules in HD-ZIPIII gain-of-function mutants, such as phb-1d orphv-1d, have aberrant integuments (Kelley et al., 2009). However,in phb-1d, WUS expression did not differ from WT (Sieber et al.,2004), suggesting that PHB has effects on integument growth thatare independent of WUS repression.

As in lateral organ primordia, CNA, PHB and PHV areexpressed in the adaxial tissue of the inner integument. Thus, itis suggested that polarity establishment is also required for innerintegument expansion (Kelley et al., 2009). The aberrant ovules incna phb phv pCNA:amiRWUS suggest that CNA, PHB and PHVpromote integument growth by other mechanisms, such asestablishing adaxial-abaxial polarity, as well as by repressingWUS. Alternatively, these aberrant ovules might be attributed tovariability in expression of the amiRWUS transgene.

HD-ZIPIII expression is not sufficient to repress WUSSeeds are formed even when either CNA, PHB or PHV isconstitutively expressed by the CaMV 35S promoter (Priggeet al., 2005), in contrast to the seedless phenotype of wus (Gross-Hardt et al., 2002), suggesting that HD-ZIPIII factors do not directlyrepress WUS expression. We drove expression of CNA under thecontrol of theWUS promoter to corroborate these results. Since HD-ZIPIII transcripts are post-transcriptionally targeted for degradationby microRNA (miR) 165/166 (Emery et al., 2003), we used CNA-δmiRNA, in which the miRNA binding site is modified to beinsensitive to miR165/166, in addition to WT CNA.

Both in pWUS:CNA-δmiRNA (Fig. 2L,O) and pWUS:CNA(Fig. 2M,P), ovules did not obviously differ from those of WT(Fig. 2K,N), suggesting that CNA requires other factors to regulateWUS in the chalaza.

Pattern of cytokinin regulation is not altered in cna phb phvWUS expression extends into the chalazal domain when cytokininis externally applied to the gynoecium (Bencivenga et al., 2012).Therefore, misexpression ofWUS in cna phb phv might result fromcytokinin upregulation. We compared cytokinin responses betweenWT and cna phb phv ovules using a TCS:GFP marker (Müller andSheen, 2008).

In WT ovules, cytokinin response is observed in the chalazaand funiculus at stage 2-III and later (Fig. 3A,B) (Bencivengaet al., 2012). The same pattern is observed in cna phb phv ovules withaberrant (Fig. 3C,D) ornormal (Fig. 3E,F) integuments, but expressionlevels are reduced, compared with WT (Fig. 3G). These data indicatethatCNA,PHB andPHV regulationofWUSexpression is not bymeansof cytokinin upregulation. In addition, ovule morphology is notaffected when cytokinin degradation is promoted by constitutiveexpression of Cytokinin oxidase/dehydrogenase genes (Werner et al.,2003), supporting the conclusion that the cna phb phv phenotype isindependent of cytokinin regulation.

Fig. 1. Patterning of ovules andWUS expression in cna phb phv. (A) Threedevelopmental domains in Arabidopsis ovules. (B-E) WUS expression in cnaphb phv at stage 2-I (B) and stage 2-III (C-E). (E) Negative control hybridizedwith sense probe. f, funiculus; n, nucellus; ii, inner integument; oi, outerintegument. Scale bars: 25 μm.

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bel1 and cna phb phv exhibit synergistic phenotypesIn bel1 an amorphous structure forms in place of the integumentsand can convert to a carpelloid organ (e.g. Robinson-Beerset al., 1992; Ray et al., 1994). In bel1 ovules WUS expressionis shifted downward into the boundary region between thenucellus and chalaza (Bencivenga et al., 2012) (Fig. S2). Thus,we crossed cna phb phv with bel1 to evaluate the interaction ofthe genes.

bel1 cna phb phv ovules form the amorphous structure seenin bel1, but the primary ovulate axis was observed to branchthrough the formation of ectopic ovule primordia (Fig. 3H,I). Theseextra primordia were formed directly on the funiculus below thechalaza, in contrast to the extra putative nucelli of bel1, whichare born on the adaxial side of the amorphous organ (Robinson-Beers et al., 1992). The ectopic ovules form amorphous organstypical of bel1 mutants in place of integuments (Fig. 3I). Formation

Fig. 2. Phenotypes of cna phb phv, cna phb phv pCNA:amiRWUS, pWUS:CNA-δmiRNA and pWUS:CNA. (A,B) Relative expression ofWUS quantified byqRT-PCR with three biological replicates (A), and percentage of normal ovules per gynoecium (n=15) (B). Three independent lines were examined. **P

of ectopic ovules is preceded by nodes of ectopicWUS expression inthe funiculus (Fig. 3J,K). WUS is expressed in the nucellus andchalaza of bel1 cna phb phvovules (Fig. 3J), as in cna phb phvovules(Fig. 1B,C). However, stronger expression was detected in thenucelli of the ectopic ovules at the same stage in their development(Fig. 3J). As the amorphous organ enlarged, WUS expressiondecreased in the nucellus and chalaza of the primary ovulate axis,whereas expression persisted in the ectopic ovules (Fig. 3K).The ovulate axis does not branch in cna phb phv (Kelley et al.,

2009) nor in bel1 (Robinson-Beers et al., 1992). Consistent with thenovel phenotype of bel1 cna phb phv, theWUS expression pattern inthis quadruple mutant is different from that observed in bel1 (Fig. S2)or in cna phb phv (Fig. 1B,C). These synergistic phenotypes suggestthat CNA, PHB and PHV act in a different pathway from BEL1 toregulate WUS. The combination of the two classes of mutationsappears to allow forWUS expression further down the ovule axis thanis observed in either single class. The formation of ectopic ovulesfrom the funiculus implies that cells in the funiculus manifestplacenta-like properties in the quadruple mutant.

CNA, PHB and PHV establish the boundary between nucellusand chalaza in Arabidopsis ovulesWe show that CNA, PHB and PHV play a major role in the negativeregulation ofWUS in the chalaza. This regulation contributes to theestablishment of the boundary between the nucellus and chalaza andpromotes the proper development of two integuments. CNA, PHBand PHV repress WUS expression independently from BEL1 orcytokinin upregulation (Fig. 4A).Integument growth is normal in cna, phb and phv single mutants

(Kelley et al., 2009), suggesting that they redundantly repress WUS

expression. In stage 2 ovules, expression of CNA, PHB and PHVdoes not completely overlap (Kelley et al., 2009; Sieber et al., 2004)(Fig. 4B), whereas WUS is misexpressed throughout the chalaza incna phb phv, a much broader area than the sum of the CNA, PHBand PHV expression areas. This discrepancy would imply thatCNA,PHB and PHV regulate the expression of WUS through theirrepression of a diffusing factor or an action of the factor (X inFig. 4). Auxin is a possible candidate for this factor because it ispredicted to flow through the chalaza (Bencivenga et al., 2012;Kelley et al., 2012), and alterations in the pattern of auxin responsecorrelate with ectopic WUS expression seen in bel1 mutants(Bencivenga et al., 2012). Alternatively, CNA or PHV might havebroader expression areas in stage I ovules, as actually reported forPHB (Sieber et al., 2004), and failure in suppression at this stagecould affect the expression patterns of WUS at subsequent stages.

The striking synergistic branched ovule phenotype observed inbel1 cna phv phb mutants implies that the two classes of genes actin relative independence in their suppression of WUS activityoutside of the nucellus. Angiosperm ovules are hypothesized to behomologous to the cupulate organs of some extinct gymnosperms,each of which were born on the apices of a branched axis (e.g.Doyle, 2006), but such branched structures are not observed inextant angiosperms. If this hypothesis is correct, then BEL1 andHD-ZIPIIIs could have played a role in the evolution of theunbranched ovule form seen in all extant angiosperms.

MATERIALS AND METHODSGrowth conditions and plant materialsArabidopsis plants were grown under continuous light at 23°C on soil. Seedswere sourced as described in the supplementary Materials and Methods.

Fig. 3. Cytokinin responses in cna phb phv and phenotypes of bel1 cna phb phv. (A,C,E) DIC images of stage 2-III ovules. (B,D,F) GFP expression fromthe TCS:GFP transgene. (A,B)WT (Col). (C,D) cna phb phvovulewith aberrant integuments. (E,F) cna phb phvwith normal integuments. (G) Relative expressionofGFP quantified by qRT-PCRwith three biological replicates. **P

Construction of transgenic linesThe amiRWUS fragment was synthesized following Web MicroRNADesigner 3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). amiRWUSbinds 607 to 627 nucleotides ofWUS (Fig. S1). amiRWUS, as well as pCNA(−4046 to −319), were inserted into pMLBarT (Fig. S1) using the GeneArtSeamless PLUS Cloning and Assembly Kit (Life Technologies). bel1-6/+cna-2 phb-13 phv-11 er-2 plants were transformed with the construct by thefloral dip method (Clough and Bent, 1998).

To generate pWUS:CNA-δmiRNA or pWUS:CNA, theWUS promoter andCNA cDNA sequences were obtained from Col-0 genomic DNA or cDNAby PCR, respectively. CNA-δmiRNA sequence was synthesized by overlapPCR as previously described (Emery et al., 2003). These fragments werecloned into pMLBarT as described above.

Further details of constructs and genotyping are given in thesupplementary Materials and Methods and Table S1.

In situ hybridization and GUS stainingFixation, embedding of tissue and in situ hybridization were performed aspreviously described (Mayer et al., 1998). GUS staining of pWUS>>uidAplants is described in the supplementary Materials and Methods.

MicroscopyFor scanning electron microscopy (SEM), ovules were fixed (McAbee et al.,2006) or epoxy molds were made (Williams et al., 1987). Fluorescenceimages of GFP were taken with excitation and emission wavelengths of470/20 nm and 505-530 nm, respectively, using an Axio Scope 2 Plus(Carl Zeiss).

qRT-PCR analysisTotal RNAs were extracted from gynoecia containing stage 2 ovules andcontaminating DNA was digested with DNase. qRT-PCR analyses wereperformed using the One Step SYBR PrimeScript RT-PCR Kit (Takara).WUS and GFP expression levels were normalized to those of PP2AA3(At1g13320) (Czechowski et al., 2005). For further details, see thesupplementary Materials and Methods.

AcknowledgementsWe thank John Bowman, Steven Clark, Shinobu Takada and NottinghamArabidopsis Stock Centre (NASC) for seeds; and Debra Skinner and Marissa Simonfor helpful comments.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsT.Y. and C.S.G. designed the study. T.Y. and Y.S. performed most experiments.K.H. and K.N. made constructs. T.Y. and C.S.G. wrote the manuscript. All authorscommented on the manuscript.

FundingSupported by a Japan Society for the Promotion of Science (JSPS) grant (Kakenhi)[24570098 to T.Y.]; and a US National Science Foundation (NSF) grant[IOS1354014 to C.S.G.].

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.129833/-/DC1

ReferencesBalasubramanian, S. and Schneitz, K. (2000). NOZZLE regulates proximal- distalpattern formation, cell proliferation and early sporogenesis during ovuledevelopment in Arabidopsis thaliana. Development 127, 4227-4238.

Bencivenga, S., Simonini, S., Benková, E. and Colomboa, L. (2012). Thetranscription factors BEL1 and SPL are required for cytokinin and auxin signalingduring ovule development in Arabidopsis. Plant Cell 24, 2886-2897.

Brambilla, V., Battaglia, R., Colombo, M., Masiero, S., Bencivenga, S., Kater,M. M. and Colombo, L. (2007). Genetic and molecular interactions between

BELL1 and MADS box factors support ovule development in Arabidopsis. PlantCell 19, 2544-2556.

Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K. and Scheible, W.-R. (2005).Genome-wide identification and testing of superior reference genes for transcriptnormalization in Arabidopsis. Plant Physiol. 139, 5-17.

Doyle, J. A. (2006). Seed ferns and the origin of angiosperms. J. Torrey Bot. Soc.133, 169-209.

Emery, J. F., Floyd, S. K., Alvarez, J., Eshed, Y., Hawker, N. P., Izhaki, A., Baum,S. F. and Bowman, J. L. (2003). Radial patterning of Arabidopsis shoots by classIII HD-ZIP and KANADI genes. Curr. Biol. 13, 1768-1774.

Green, K. A., Prigge, M. J., Katzman, R. B. and Clark, S. E. (2005). CORONA, amember of the Class III Homeodomain Leucine Zipper gene family in Arabidopsis,regulates stem cell specification and organogenesis. Plant Cell 17, 691-704.

Gross-Hardt, R., Lenhard, M. and Laux, T. (2002). WUSCHEL signaling functionsin interregional communication during Arabidopsis ovule development. GenesDev. 16, 1129-1138.

Kelley, D. R., Skinner, D. J. and Gasser, C. S. (2009). Roles of polaritydeterminants in ovule development. Plant J. 57, 1054-1064.

Kelley, D. R., Arreola, A., Gallagher, T. L. andGasser, C. S. (2012). ETTIN (ARF3)physically interacts with KANADI proteins to form a functional complex essentialfor integument development and polarity determination in Arabidopsis.Development 139, 1105-1109.

Landau, U., Asis, L. andWilliams, L. E. (2015). TheERECTA,CLAVATA and classIII HD-ZIP pathways display synergistic interactions in regulating floral meristemactivities. PLoS ONE 10, e0125408.

Lee, C. and Clark, S. E. (2015). AWUSCHEL-independent stem cell specificationpathway is repressed by PHB, PHV and CNA in Arabidopsis. PLoS ONE 10,e0126006.

Mandel, T., Moreau, F., Kutsher, Y., Fletcher, J. C., Carles, C. C. and Williams,L. E. (2014). The ERECTA receptor kinase regulates Arabidopsis shoot apicalmeristem size, phyllotaxy and floral meristem identity. Development 141,830-841.

Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M., Jürgens, G. and Laux, T.(1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shootmeristem. Cell 95, 805-815.

McAbee, J. M., Hill, T. A., Skinner, D. J., Izhaki, A., Hauser, B. A., Meister, R. J.,Venugopala Reddy, G., Meyerowitz, E. M., Bowman, J. L. and Gasser, C. S.(2006). ABERRANT TESTA SHAPE encodes a KANADI family member, linkingpolarity determination to separation and growth of Arabidopsis ovule integuments.Plant J. 46, 522-531.

McConnell, J. R., Emery, J., Eshed, Y., Bao, N., Bowman, J. and Barton, M. K.(2001). Role ofPHABULOSA andPHAVOLUTA in determining radial patterning inshoots. Nature 411, 709-713.

Müller, B. and Sheen, J. (2008). Cytokinin and auxin interaction in root stem-cellspecification during early embryogenesis. Nature 453, 1094-1098.

Prigge, M. J., Otsuga, D., Alonso, J. M., Ecker, J. R., Drews, G. N. and Clark,S. E. (2005). Class III homeodomain-leucine zipper gene family members haveoverlapping, antagonistic, and distinct roles in Arabidopsis development. PlantCell 17, 61-76.

Ray, A., Robinson-Beers, K., Ray, S., Baker, S. C., Lang, J. D., Preuss, D.,Milligan, S. B. and Gasser, C. S. (1994). The Arabidopsis floral homeotic geneBELL (BEL1) controls ovule development through negative regulation ofAGAMOUS gene (AG). Proc. Natl. Acad. Sci. USA 91, 5761-5765.

Robinson-Beers, K., Pruitt, R. E. and Gasser, C. S. (1992). Ovule development inwild-type Arabidopsis and two female-sterile mutants. Plant Cell 4, 1237-1249.

Schneitz, K., Hulskamp, M. and Pruitt, R. E. (1995). Wild-type ovule developmentin Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue.Plant J. 7, 731-749.

Sieber, P., Gheyselinck, J., Gross-Hardt, R., Laux, T., Grossniklaus, U. andSchneitz, K. (2004). Pattern formation during early ovule development inArabidopsis thaliana. Dev. Biol. 273, 321-334.

Tucker, M. R., Hinze, A., Tucker, E. J., Takada, S., Jürgens, G. and Laux, T.(2008). Vascular signalling mediated by ZWILLE potentiates WUSCHEL functionduring shoot meristem stem cell development in the Arabidopsis embryo.Development 135, 2839-2843.

Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H. andSchmülling, T. (2003). Cytokinin-deficient transgenic Arabidopsis plants showmultiple developmental alterations indicating opposite functions of cytokinin inregulation of shoot and root meristem activity. Plant Cell 15, 2532-2550.

Williams, M. H., Vesk, M. and Mullins, M. G. (1987). Tissue preparation forscanning electron microscopy of fruit surfaces: comparison of fresh andcryopreserved specimens and replicas of banana peel. Micron. Microsc. Acta18, 27-31.

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